Regulation Of MEBO On Proliferation And Autophagy Related Genes LC3,beclin-1 And P62 Of Fibroblasts Cultured In High Glucose

Objective: High glucose culture of fibroblasts in vitro,to observe the effect of MEBO on the growth of fibroblasts cultured in high glucose.In this study,we observed the regulatory effect of MEBO on autophagy related genes LC3,beclin-1 and p62 of fibroblasts cultured in high glucose,and explored the mechanism of MEBO / MEBT in promoting diabetic chronic wound healing,so as to offer theoretical basis for clinical application.Methods: PartⅠ: The wound model of diabetic rats was established.After 11 days,the fibroblasts were isolated and cultured,and identified by immunofluorescence.Then the high glucose medium containing MEBO was prepared,and the optimal growth concentration was explored.After digestion and transmission to the third generation,the cells were randomly divided into two groups: control group and MEBO group,(1)CCK-8 method was used to detect the 12 th hour The proliferation of fibroblasts was observed in 24 hours,48 hours,72 hours and 96 hours;(2)the effects of MEBO on fibroblast migration were observed by scratch test.PartⅡ: The above fibroblasts were digested and passaged to the third passage,the cells in logarithmic phase(the third day)were divided into the following 7 groups: blank group,model group,MEBO low concentration group,MEBO medium concentration group,MEBO high concentration group,rapamycin+MEBO group and rapamycin group(Except for the blank group,the other groups were treated with high glucose medium).The results showed that:(1)immunofluorescence detection of LC3,Beclin-1 and p62 staining;(2)Autophagosome was detected by transmission electron microscope;(3)QRT PCR was used to detect the m RNA expression of LC3,Beclin-1 and p62;(4)The protein expressions of LC3,Beclin-1 and p62 were detected by Western blotting(WB).Results: PartⅠ:(1)Cell proliferation test: the OD value of MEBO group at each time point was higher than that of the control group from the 48 th hour(P<0.05),and the cell proliferation rate was the highest at the 72 th hour;(2)scratch test showed that MEBO group promoted fibroblast migration better than control group,the difference was statistically significant(P<0.05).Part Ⅱ :(1)Immunofluorescence test showed that compared with the blank group,the highest OD value of LC3 and Beclin-1 in each group was MEBO high concentration group,the lowest was blank group,and OD value of low,medium and high concentration group of MEBO increased gradually,the OD value of rapamycin+MEBO group in Beclin-1 was higher than that of rapamycin group;the highest OD value of p62 fluorescence staining was blank group,the lowest was MEBO high concentration group,MEBO was low The OD value of the medium and high concentration groups decreased gradually,and the OD value of rapamycin group was higher than that of rapamycin+MEBO group;(2)Electron microscopy showed that compared with the blank group,the number of autophagosomes in MEBO high concentration group,rapamycin group and rapamycin +MEBO group was more and more typical,and the number of autophagosomes in MEBO low,medium and high concentration groups increased gradually;(3)q RT-PCR showed that compared with the blank group,the relative expression of rapamycin+MEBO group in LC3 group and Beclin-1 group was the highest,and the expression of rapamycin+MEBO group was higher in LC3 group than that in rapamycin group;p62 The relative expression of rapamycin group was the lowest,and the expression of rapamycin decreased in MEBO group;(4)Western blotting showed that the expression of rapamycin+MEBO and MEBO in LC3 and Beclin-1 were significantly higher in each group.The expression of various factors in MEBO group increased with the increase of concentration,and the expression of rapamycin group increased after MEBO,while that of p62 eggs increased White expression is opposite.Conclusion: MEBO can increase the expression of autophagy related genes LC3 and Beclin-1 and decrease the expression of p62 in fibroblasts cultured in high glucose medium,regulate autophagy and promote fibroblast proliferation,which may be one of the mechanisms of MEBT / MEBO promoting diabetic chronic wound repair.